During 2013-14, Dr. Aravind demonstrated significant progress and effective planning and execution of several major research projects. These research projects are in the areas of molecular enzymology, signal transduction, and bacterial pathogenesis and inter-organismal conflicts using computational methods. His groupcomprised of two staff scientists, two post-doctoral fellows, and one contractorhas published numerous peer-reviewed publications in reputed scientific journals. Some highlights of Dr. Aravinds 2013 research program include the following: Dr. Aravind in collaboration with Dr. K.Ramamurthi from NCI showed how the bacterial morphogenetic protein SpoIVA is related to translation factor GTPases and how ATP hydrolysis by it drives polymerization. The assembly of static supramolecular structures is a culminating event of developmental programs. One such structure, the proteinaceous shell (called the coat) that surrounds spores of the bacterium Bacillus subtilis, is composed of about 70 different proteins and represents one of the most durable biological structures known. The coat is built atop a basement layer that contains an ATPase (SpoIVA) that forms a platform required for coat assembly. Here, they showed that SpoIVA belongs to the translation factors class of P-loop GTPases and has evolutionarily lost the ability to bind GTP; instead, it uses ATP hydrolysis to drive its self-assembly into static filaments. They demonstrated that ATP hydrolysis is required by every subunit for incorporation into the growing polymer by inducing a conformational change that drives polymerization of a nucleotide-free filament. SpoIVA therefore differs from other self-organizing polymers (dynamic cytoskeletal structures and static intermediate filaments) in that it uses ATP hydrolysis to self-assemble, not disassemble, into a static polymer. We further show that polymerization requires a critical concentration that they propose is only achieved once SpoIVA is recruited to the surface of the developing spore, thereby ensuring that SpoIVA polymerization only occurs at the correct subcellular location during spore morphogenesis. Dr. Aravind in collaboration with Dr. Anjana Raos team from UCSD uncovered a new mechanism for the regulation of the TET proteins with implications for DNA demethylation and cancer progression. The TET proteins are Fe(ii)- and &#945;-ketoglutarate-dependent dioxygenases that modify the methylation status of DNA by successively oxidizing 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, potential intermediates in the active erasure of DNA-methylation marks. They showed that IDAX (also known as CXXC4), a reported inhibitor of Wnt signaling that has been implicated in malignant renal cell carcinoma and colonic villous adenoma, regulates TET2 protein expression. Dr. Aravind showed that IDAX was originally encoded within an ancestral TET2 gene that underwent a chromosomal gene inversion during evolution, thus separating the TET2 CXXC domain from the catalytic domain. The IDAX CXXC domain binds DNA sequences containing unmethylated CpG dinucleotides, localizes to promoters and CpG islands in genomic DNA and interacts directly with the catalytic domain of TET2. Unexpectedly, IDAX expression results in caspase activation and TET2 protein downregulation, in a manner that depends on DNA binding through the IDAX CXXC domain, suggesting that IDAX recruits TET2 to DNA before degradation. IDAX depletion prevents TET2 downregulation in differentiating mouse embryonic stem cells, and short hairpin RNA against IDAX increases TET2 protein expression in the human monocytic cell line U937. Notably, they found that the expression and activity of TET3 is also regulated through its CXXC domain. Taken together, these results established the separate and linked CXXC domains of TET2 and TET3, respectively, as previously unknown regulators of caspase activation and TET enzymatic activity. Dr. Aravind in collaboration with Dr. Louis Millers group uncovered the mechanism for how the malarial parasite expresses variable surface antigens. The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. They showed that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncovered a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologs of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine. Dr. Aravind and his team discovered new components of the DNA repair system using computational analysis. The bacterial SOS response is an elaborate program for DNA repair, cell cycle regulation and adaptive mutagenesis under stress conditions. Using sensitive sequence and structure analysis, combined with contextual information derived from comparative genomics and domain architectures, he and his team identified two novel domain superfamilies in the SOS response system. They presented evidence that one of these, the SOS response associated peptidase (SRAP; Pfam: DUF159) is a novel thiol autopeptidase. Given the involvement of other autopeptidases, such as LexA and UmuD, in the SOS response, this finding suggests that multiple structurally unrelated peptidases have been recruited to this process. The second of these, the ImuB-C superfamily, is linked to the Y-family DNA polymerase-related domain in ImuB, and also occurs as a standalone protein. They presented evidence using gene neighborhood analysis that both these domains function with different mutagenic polymerases in bacteria, such as Pol IV (DinB), Pol V (UmuCD) and ImuA-ImuB-DnaE2 and also other repair systems, which either deploy Ku and an ATP-dependent ligase or a SplB-like radical SAM photolyase. We suggest that the SRAP superfamily domain functions as a DNA-associated autoproteolytic switch that recruits diverse repair enzymes upon DNA damage, whereas the ImuB-C domain performs a similar function albeit in a non-catalytic fashion. They proposed that C3Orf37, the eukaryotic member of the SRAP superfamily, which was recently shown to specifically bind DNA with 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxycytosine, is a sensor for these oxidized bases generated by the TET enzymes from methylcytosine. Hence, its autoproteolytic activity might help it act as a switch that recruits DNA repair enzymes to remove these oxidized methylcytosine species as part of the DNA demethylation pathway downstream of the TET enzymes.